Novel Epitopes of the Influenza Virus N1 Neuraminidase Targeted by Human Monoclonal Antibodies

ABSTRACT Influenza virus neuraminidase (NA)-targeting antibodies are an independent correlate of protection against influenza. Antibodies against the NA act by blocking enzymatic activity, preventing virus release and transmission. As we advance the development of improved influenza virus vaccines that incorporate standard amounts of NA antigen, it is important to identify the antigenic targets of human monoclonal antibodies (mAbs). Here, we describe escape mutants generated by serial passage of A/Netherlands/602/2009 (H1N1)pdm09 in the presence of human anti-N1 mAbs. We observed escape mutations on the head domain of the N1 protein around the enzymatic site (S364N, N369T, and R430Q) and also detected escape mutations located on the sides and bottom of the NA (N88D, N270D, and Q313K/R). This work increases our understanding of how human antibody responses target the N1 protein. IMPORTANCE As improved influenza virus vaccines are being developed, the influenza virus neuraminidase (NA) is becoming an important new target for immune responses. By identifying novel epitopes of anti-NA antibodies, we can improve vaccine design. Additionally, characterizing escape mutations in these epitopes aids in identifying NA antigenic drift in circulating viruses.

IMPORTANCE As improved influenza virus vaccines are being developed, the influenza virus neuraminidase (NA) is becoming an important new target for immune responses. By identifying novel epitopes of anti-NA antibodies, we can improve vaccine design. Additionally, characterizing escape mutations in these epitopes aids in identifying NA antigenic drift in circulating viruses. KEYWORDS N1, influenza, mAbs, neuraminidase I nfluenza viruses cause seasonal epidemics and, occasionally, global pandemics that lead to significant morbidity and mortality worldwide (1,2). They are a member of the family Orthomyxoviridae and contain a segmented, negative-sense RNA genome. Two of the genomic segments encode the glycoproteins present on the viral surface, the hemagglutinin (HA) and the neuraminidase (NA) (3,4). The HA of influenza viruses, which is responsible for receptor binding and viral entry, has been largely credited as the immunodominant target of the antibody response after vaccination and natural infection (3)(4)(5). The NA acts as a sialidase, removing terminal sialic acids and allowing viral egress and spread. It has recently become appreciated as an additional important target of anti-influenza virus immunity (6)(7)(8)(9). To function properly, the NA must be present on the viral surface as a homotetramer (10)(11)(12).
Seasonal influenza virus vaccines are the first line of defense against infection (13). Typically, these vaccines are standardized based on the HA content but have varying NA content with unknown structural integrity (14,15). In addition, seasonal vaccines can have varying effectiveness from 20% to 60% in a given year (16). Low vaccine effectiveness can be largely attributed to the antigenic variability of the HA vaccine component compared
Escape mutant viruses are resistant to binding, NAI, and neutralization activities of mAbs. We next used the mAbs to evaluate the impact of each NA mutation on antibody binding, NAI, and neutralization activities. Using immunofluorescence assays, we identified that the N270D mutation impacted most of the antibodies in our panel, including those that produced other EMVs ( Fig. 2A). The N88D mutation affected the binding of only the mAb that caused the mutation, 1000-3C05. Q313K impacted the binding of the mAb that caused that mutation (294-16-009-A-1C02) and mAb 294-16-009-A-1D05, while Q313R impacted the binding of only 294-16-009-A-1C02. S364N and the double mutation S364N/N369T affected the binding of EM-2E01 and 1000-1D05 ( Fig. 2A). All mAbs retained binding to the R430Q EMV. Additionally, recombinant NA (rNA) proteins containing each EMV mutation were generated, and mAb binding was assessed using an enzyme-linked immunosorbent assay (ELISA). We observed similar changes in binding toward rNAs, with N270D impacting the binding of a majority of mAbs in the panel (Fig. 2B). N88D impacted the binding of only 1000-3C05. Interestingly, Q313K and Q313R rNAs exhibited altered binding with EM-2E01 and 1000-1D05, along with 294-16-009-A-1C02 and 294-16-009-A-1D05. Of note, reduced binding has also been reported for 1000-1D05 for an N309S mutant, which is a position close to Q313 (14). We observed a slight loss of binding of EM-2E01 to S364N rNA; however, the N369T and the double mutant S364N/N369T rNAs maintained mAb binding. Both R430Q and G454D had little impact on overall mAb binding, although reduced binding was observed for 294-16-009-A-1C02 and 294-16-009-A-1D05.
To determine changes in NAI activity, we performed ELLAs with each EMV in the presence of each mAb. Additionally, we performed NAI assays using rNA proteins. While 1000-3C05 has no NAI activity, we still assessed if the mutation that it potentially caused, N88D, impacted any other antibodies in the panel. We found no significant changes in the NAI activity of any of the mAbs tested with N88D ( Fig. 3A and B and Table 3). We found that the N270D EMV exhibited complete escape from 1000-3B04 and 294-16-009-A-1C02, along with resistance to 1000-3B06 (42-fold increase in the NAI IC 50 ) and 300-16-005-G-2A04 (30-fold change in the NAI IC 50 ) ( Fig. 3A and Table 3). The N270D rNA exhibited a similar phenotype, with complete escape from 1000-3B04, 1000-3B06, and 294-16-009-A-1C02 along with resistance to 300-16-005-G-2A04 (168-fold change in the NAI IC 50 ) (Fig. 3B). The Q313K EMV and rNA became resistant to the  Table 3). S364N and S364N/N369T EMVs completely escaped from  The darker gray subunit has residues identified in previous publications and the NA active site (in white). Murine epitopes are illustrated in blue (27), magenta (28), and yellow (29). Human epitopes are indicated in orange (14), cyan (31), green (30), and indigo (32). Mutations identified in the EMVs used for this study are highlighted in red and identified using arrows. Views from the top (A), side (B), and bottom (C) of the NA are depicted.
Characterization of escape mutant viruses in vivo. Evaluating the in vivo fitness of each EMV is important to determine if natural isolates that acquire these mutations will have a probability of showing increased or decreased fitness. However, since the EMVs created through passaging acquired mutations outside the NA, likely including cell culture-adaptive mutations, it is not possible with this set of viruses to determine which mutations directly impact fitness. Nevertheless, we still went ahead with these experiments because the outcome can at least indicate if any of the NA mutations severely attenuate the EMVs in vivo. We determined the mouse 50% lethal dose (mLD 50 ) of each EMV to assess fitness changes in vivo. Since both Q313R EMV and S364N EMV had similar changes in binding, NAI, and neutralization activities to Q313K and S364N/N369T EMVs, only one was chosen for mLD 50 experiments. Wild-type A/ Netherlands/602/2009 (H1N1)pdm09 virus had an mLD 50 of 1 PFU (Table 5). A majority of EMVs had mLD 50 values similar to that of the wild-type virus, including the N88D, N270D, Q313K, and S364N/N369T EMVs ( Table 5). The R430Q EMV had a moderate (45fold) increase in the mLD 50 (Table 5). However, we also noted that this EMV contained a unique HA mutation, K226M. As explained above, this experiment has significant caveats. However, it suggests that none of the mutations detected severely attenuated the EMVs. This is different than what has been observed with similar EMVs generated against stalk-binding antibodies where severe attenuation in vivo was detected (35).

DISCUSSION
Our study has identified novel epitopes on the N1 targeted by human mAbs. Only 2 of the escape mutations detailed here have been previously reported, N270 and N369, indicating that these 2 residues are frequently targeted epitopes of human mAbs (14,27,30). Interestingly, a majority of the 2017-2018 isolates contained N270K and N369K mutations, which further emphasizes their importance for mAb binding and NAI activity. The remaining mutations, N88D, Q313K/R, and S364N, are part of newly identified mAb epitopes.
The mutation N88D, a critical residue for 1000-3C05, is located very close to the NA head-stalk interface. mAb 1000-3C05 does not exhibit NAI activity and is poorly neutralizing; however, previous reports have noted that it is protective in vivo, is cross-reactive with several human N1s (pre-and postpandemic), and can utilize Fc effector functions (14,31). The position of N88 on the protein may explain why 1000-3C05 is NAI inactive as this mAb does not interfere with the enzymatic site of the NA.
Mutations at Q313 were necessary for the evasion of 294-16-009-A-1C02 during escape mutagenesis. Once the EMV was identified, we noted that this residue was also important for another mAb in our panel, namely, 1000-1D05. This residue is located on the side of the NA, outside any previously defined antigenic regions. We noticed that the Q313K mutation had a slightly stronger effect on mAb escape than Q313R. This may be best explained by amino acid biochemistry. Lysine has a lesser degree of freedom to form electrostatic interactions as compared to arginine and this 'rigidity' could lead to stronger interruption of antibody-antigen interactions.
We identified two separate EMVs containing the mutation S364N. When comparing escape phenotypes, both the S364N and S364N/N369T EMVs completely escaped the mAb EM-2E01, became resistant to the neutralization of 1000-1D05, and remained sensitive to the remaining mAbs in the panel. These data suggest that S364N is sufficient for escape from EM-2E01 and that N369T is not critical for mAb escape, which was confirmed with recombinant NA carrying either S364N or N369T. Furthermore, the S364N mutation alone introduces an N-linked glycosylation site, which is likely responsible for blocking mAb activity. This is interesting because many recent isolates contain mutations at N369, like the A/New York/PV01575/2018 isolate used in this study, but S364 is highly conserved.
To truly understand how these mutations impact NA antigenicity, it would be important for future studies to test how human sera inhibit the neuraminidase activity of our EMVs. Nevertheless, our findings can already aid in annual vaccine strain selection since they can help to identify amino acid changes in circulating strains that may lead to antigenic drift of the NA. In addition, our findings can also inform the design of NAbased vaccines (35,36). Specifically, they indicate which amino acids are important for interactions with human antibodies. This information helps in the design of immunogens in which epitopes that include these amino acid positions are displayed correctly. Conversely, it can also help in the design of antigens in which the targeting of these amino acid positions is avoided since they are prone to change.
Anti-NA antibodies can inhibit NA through several mechanisms. They can bind directly to the active site, thereby blocking the access of the substrate to it. They can also bind further away and block interactions between the active site and substrate through steric hindrance. Finally, it could also be hypothesized that antibodies may bind far away from the enzymatic site, triggering allosteric changes that incapacitate the active site. As described previously by Chen et al. (14), EM-2E01 inhibited the NA in both an NAI assay using sialic acid attached to an N-linked glycan on large, bulky fetuin as the substrate as well as an NAI assay using a small-molecule substrate. This suggests that the mAb binds directly to the active site. The fact that EM-2E01 drove escape mutation S364N, which is close to the active side, supports this hypothesis. All other mAbs used here (with the exception of 300-16-005-G-2A04) exhibit activity only in NAI assays with fetuin but not in assays with a small-molecule substrate (14), suggesting binding outside the enzymatic site and inhibition via steric hindrance. Again, this is supported by the escape mutations that we found for these mAbs.
In addition to mutations in the NA gene, most EMVs also contained mutations R62K, D239G, and R240Q in their HA. Both D239G and R240Q have been previously reported to increase virus growth in vitro (37,38). The mutation R62K has not yet been fully characterized; however, it has been observed in natural isolates and may be involved in HA stability (39). Both the N270D and Q313K EMVs shared the HA mutation K136N, which has not yet been described in the literature. The R430Q EMV acquired a unique HA mutation, K226M, which has also not been previously characterized. Based on our in vivo fitness data, it appears that this HA mutation could have a slight impact on viral fitness. Importantly, the R62K, K163N, D239G, and R240Q mutations were also found in one of the control viruses that was passaged without pressure from NA-specific mAbs, suggesting that these mutations represent cell culture-adaptive mutations. The second control virus contained an HA mutation, E391G, which was also present in the A/New York/PV01575/2018 isolate. The NP mutation S50N present in both the Q313K EMV and the R430Q EMV has been identified as a mutation that does not impact polymerase activity in vitro, leading us to conclude that this mutation does not have an impact on the viral fitness observed (40). The PA mutations identified in these viruses, V100L and V407I, have not yet been characterized in the literature. Additionally, the NP mutation E372D and the M1 mutation E204D, which were identified only in the N88D The only statement that can be made about the escape mutants is that according to in vivo data, the mutations in the NA did not result in a strongly attenuated phenotype. This is in contrast to what we have observed with the same virus strain when we used a similar approach to map anti-HA stalk mAbs (34). Escape from antistalk mAbs caused a strong attenuation phenotype in many EMVs. Combined with data from previous reports, we can conclude that human antibodies are targeting more than just the enzymatic site (14,(30)(31)(32). Figure 1 illustrates where the EMV mutations, along with others discussed here, are located on the NA. Aside from overlaps at positions 248, 249, 270, 273, 309, 369, 451, and 456, the epitopes for murine mAbs are unique compared to what has been observed for human mAbs. This highlights why it is important to evaluate antigenic sites using human monoclonal antibodies to increase our understanding of how the N1 is being targeted by our immune responses. A/Netherlands/602/2009 (H1N1)pdm09 was grown in our laboratory by injection of 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs (Charles River Laboratories) and incubation at 37°C for 2 days. All mAbs were identified and isolated previously and provided by Patrick Wilson (14). They were expressed in our laboratory using the Expi293 transfection kit according to the manufacturer's instructions (Thermo Fisher). mAbs were purified by gravity flow with protein G-Sepharose-packed columns and concentrated as described previously (41).

MATERIALS AND METHODS
Escape mutant generation. Escape mutant viruses were generated using the H1N1 virus A/ Netherlands/602/2009 (H1N1)pdm09 as the maternal strain. MDCK cells were plated at 6 Â 10 5 cells/mL in a 12-well, sterile cell culture plate and incubated overnight at 37°C with 5% CO 2 . The following day, virus was diluted to an MOI of 0.01 (1 Â 10 3 PFU) in 1Â minimal essential medium (MEM) (10% 10Â MEM [Gibco], 2 mM L-glutamine [Gibco], 0.1% sodium bicarbonate [Gibco], 10 mM HEPES, 1-U/mL penicillin-1-mg/mL streptomycin solution, and 0.2% bovine serum albumin [BSA]) supplemented with 1 mg/mL tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin. Antibodies were then added to the virus at a concentration that was 0.25 times the neuraminidase inhibition (NAI) 50% inhibitory concentration (IC 50 ). For the mAbs without NAI activity (1000-3C05 and 294-16-009-A-1D05), the plaque reduction neutralization assay (PRNA) IC 50 was used instead. The virus-antibody mixture was incubated with shaking for 1 h at room temperature (RT). MDCK cells were washed with 1Â phosphate-buffered saline (PBS; Gibco), and the mixture was then added to the cells. They were then incubated at 37°C with 5% CO 2 for 2 days. The supernatant was collected and stored at 280°C until further use. For subsequent passages, MDCK cells were plated as described above, infected with a 1:10 dilution of the previous passage in 1Â MEM with TPCK-treated trypsin (1 mg/mL), and incubated for 40 min at 37°C with 5% CO 2 . In the meantime, mAb was diluted in 1Â MEM with TPCK-treated trypsin to a concentration that was doubled from the previous passage (passage 1 was 0.25Â IC 50 , passage 2 was 0.5Â, and so on). After 40 min, diluted mAb was added to the virus-infected cells and left for 2 days at 37°C with 5% CO 2 . The cell culture supernatant was screened for escape mutant viruses by plaque assays with 128Â IC 50 of mAb present in the agarose overlay. Individual plaques (3 to 6 per EMV) were chosen and propagated in SPF eggs for 2 days at 37°C as described above.
RNA isolation and deep sequencing. RNA was isolated from egg allantoic fluid using the E.Z.N.A. viral RNA extraction kit (Omega Bio-Tek) according to the manufacturer's instructions and then underwent next-generation sequencing. Sequences were assembled using a pipeline designed at the Icahn School of Medicine at Mount Sinai as described previously (42). To identify point mutations, full-length coding sequences were compared to the sequenced wild-type A/Netherlands/602/2009 (H1N1)pdm09 strain used for escape mutagenesis. All reported mutations were numbered from the methionine of each protein.
Plaque assay. Plaque assays were performed using a standard protocol. MDCK cells were seeded 24 h previously at 8 Â 10 5 cells/mL in a sterile, 12-well plate and incubated overnight at 37°C with 5% CO 2 . Next, virus samples were serially diluted in 1Â MEM from 10 21 to 10 26 . MDCK cells were washed with 1Â PBS and then infected with 200 mL of each virus dilution. Virus was incubated for 40 min at 37°C with 5% CO 2 , with rocking every 10 min. Afterward, virus was aspirated and immediately replaced with 1 mL of an agarose overlay containing 2Â MEM, 0.1% diethylaminoethyl (DEAE)-dextran, 1 mg/mL TPCK-treated trypsin, and 0.64% Oxoid agarose. Plates were incubated for 2 days at 37°C with 5% CO 2 . Cells were then fixed using a 3.7% solution of paraformaldehyde (PFA) and incubated at 4°C overnight.
For plaque visualization, the overlay was removed, and cells were stained with a solution containing 20% methanol and 0.5% crystal violet.
ELISA. Recombinant NA proteins of A/California/7/2009 (H1N1) with mutations were produced using the well-established baculovirus expression system, which has been described in detail previously (43). Immulon 4HBX 96-well microtiter plates (Thermo Fisher Scientific) were coated overnight at 4°C with recombinant proteins (100 ng/well) in PBS (pH 7.4). The well contents were discarded and blocked with 200 mL of 3% nonfat milk (American Bio) in PBS containing 0.1% Tween 20 (PBST) for 1 h at RT. After blocking, 50 mL of monoclonal antibodies diluted (starting at an Ig concentration of 10 mg/mL and serially diluted 3-fold) with 1% nonfat milk in PBST was added to each well for reaction at RT for 2 h. After washing with PBST three times, 50 mL of horseradish peroxidase (HRP)-labeled goat anti-human IgG (H1L) cross-adsorbed secondary antibody (Thermo Fisher Scientific) diluted 2,500-fold with 1% nonfat milk in PBST was added to each well, and the mixture was incubated at RT for 1 h. After washing with PBST three times, 100 mL of SigmaFast o-phenylenediamine dihydrochloride (OPD) substrate solution (Sigma-Aldrich) was added to each well for the reaction at RT for 10 min. The reaction was stopped by the addition of 50 mL of 3 M hydrochloric acid (HCl). The optical density at 490 nm was measured using a Synergy 4 plate reader (BioTek). The area under the curve (AUC) was calculated in GraphPad Prism 9. Percent binding was determined by comparing the wild-type rNA AUC to mutant rNA AUC values.
Immunofluorescence. MDCK cells were plated at 3 Â 10 5 cells/mL in a sterile, 96-well plate and incubated overnight at 37°C with 5% CO 2 . The following day, cells were checked for .99% confluence and washed with 1Â PBS. Virus was diluted to an MOI of 5 in 1Â MEM and added to each well (100 mL/well). Plates were incubated overnight at 37°C with 5% CO 2 . The following day, cells were fixed using 200 mL of 3.7% PFA and incubated overnight at 4°C. Next, the PFA was removed, and cells were blocked with 1Â PBS containing 3% nonfat milk (American Bio) for 1 h at room temperature. The blocking solution was then removed and replaced with 1% nonfat milk. Primary mAbs were diluted to 300 mg in 1Â PBS and added to the 1% milk at a 1:10 dilution, for a final concentration of 30 mg per well. Primary antibodies were incubated with shaking for 1 h at room temperature. Plates were then washed 3 times with 1Â PBS. The secondary antibody Alexa Fluor 488 goat anti-human IgG(H1L) (Invitrogen) was diluted to 1:500 in 1% milk and added to the plates, and the mixture was incubated for 1 h at room temperature in the dark with shaking. The plates were then washed 3 times with 1Â PBS. To prevent cells from drying out, 50 mL of 1Â PBS was added to each well. Plates were visualized using the Celigo S adherent cell cytometer (Nexcelom Bioscience) with the 2-channel target 112 (merge) setting. The exposure time, gain, and focus (set using image-based autofocus with the 488-nm signal as the target) for the 96-well plate were automatically determined by the machine. Fluorescence was calculated using the default analysis settings, and percent fluorescence was determined based on the wild-type signal. We performed 2 independent assays; however, representative images from only 1 assay are shown here. The mAb CR9114 was included as a positive control to illustrate that all viruses had similar infectivities. This mAb is a pan-influenza A virus (IAV) HA stalk mAb (44).
Enzyme-linked lectin assay. Flat-bottom Immulon 4HBX microtiter plates (Thermo Scientific) were coated with 25 mg/mL of fetuin (Sigma) diluted in 1Â PBS, at 100 mL per well and incubated overnight at 4°C. The next day, viruses were serially diluted (3-fold) in sample diluent buffer (1Â PBS with 0.5 mM MgCl 2 , 0.9 mM CaCl 2 , 1% BSA, and 0.5% Tween 20) in a sterile 96-well plate. Once diluted, an additional 1:1 ratio of the sample diluent was added to the plate. This mixture was incubated for 1 h at room temperature with shaking. After 1 h, the fetuin-coated plates were washed 3 times with PBST using the AquaMax 3000 automated plate washer. Diluted virus was immediately added to the plates, and the plates were then incubated at 37°C with 5% CO 2 for 18 h (overnight). The following day, plates were washed 6 times with PBST. Peanut agglutinin (PNA; Sigma) was diluted to 5 mg/mL in conjugate diluent buffer (1Â PBS with 0.5 mM MgCl 2 , 0.9 mM CaCl 2 , and 1% BSA) and added to the washed plates. This mixture was incubated for 2 h in the dark at room temperature. The PNA was then removed, and plates were washed 3 times with PBST. SigmaFast OPD (Sigma) was diluted in water. OPD was added at 100 mL per well, and the mixture was incubated for 3 min at room temperature. The reaction was stopped by adding 50 mL of 3 M hydrochloric acid, and the absorbance (at 490 nm) was then immediately determined using a Synergy H1 hybrid multimode microplate reader (BioTek). Prism 7.0 was used to determine the effective concentration of each virus that would yield detectable NA activity. Each enzyme-linked lectin assay (ELLA) was done in triplicate.
Neuraminidase inhibition assay. To determine the NAI activity of each mAb, flat-bottom Immulon 4HBX microtiter plates (Thermo Scientific) were coated with 25 mg/mL fetuin (Sigma) diluted in 1Â PBS and incubated overnight at 4°C. The following day, antibodies were diluted in sample diluent buffer and then serially diluted 1:3 in a sterile 96-well plate. Virus or rNA was diluted to 2 times the effective concentration determined by an ELLA and added to mAbs at a 1:1 ratio. This mixture was incubated for 1 h at room temperature with shaking. The fetuin-coated plates were washed 3 times with PBST as described above. Virus-mAb dilutions were transferred to the fetuin-coated plates and incubated at 37°C, with 5% CO 2 , for 18 h (overnight). The following day, we performed the ELLA procedure as described above. The IC 50 and 95% confidence intervals were determined using a nonlinear regression [log(inhibitor) versus response-variable slope (4 parameters)] in Prism 7.0. Each NAI assay was performed in duplicate. Significance between IC 50 values for each EMV and the irrelevant IgG control virus was calculated using 2-way analysis of variance (ANOVA). The fold change in the mAb NAI IC 50 was calculated by dividing the EMV IC 50 by the IC 50 of irrelevant IgG control virus B. Fold changes in NAI IC 50 s for rNAs were calculated by dividing the rNA IC 50 by the wild-type rNA IC 50 .
Plaque reduction neutralization assay. MDCK cells were plated at 8 Â 10 5 cells/mL in 12-well plates. The following day, mAbs were diluted to 100 mg/mL in 300 mL 1Â MEM and serially diluted 1:5 in 1Â MEM to a final concentration of 0.032 mg/mL. Each virus was then diluted in 1Â MEM to 1 Â 10 3 PFU, and 50 mL was added to each antibody dilution. This virus-mAb mixture was incubated for 1 h at room temperature with shaking. Afterward, MDCK cells were washed with 1Â PBS and then immediately infected with 200 mL per well of the virus-mAb mixture. The plates were incubated for 40 min at 37°C with 5% CO 2 , rocking every 10 min. In the meantime, the overlay was prepared. Antibodies were diluted to 100 mg/mL in 625 mL of 2Â MEM and then serially diluted as described above. Next, a solution containing 1Â DEAE-dextran and 1 mg/ mL of TPCK-treated trypsin in sterile water for injection (Gibco) was added at 180 mL to each antibody dilution. When the infection finished, 360 mL of 2% Oxoid agarose was added to the overlay mixture in small batches to prevent solidification before being transferred to cells. The inoculum was removed and immediately replaced with the overlay so that the mAb dilution in the overlay was the same as the concentration in the inoculum. The plates were then incubated at 37°C, with 5% CO 2 , for 2 days. Cells were fixed and stained as described above. When determining plaque numbers per well, all plaques present were counted regardless of their size. The IC 50 and 95% confidence intervals were determined using a nonlinear regression [log(inhibitor) versus response-variable slope (4 parameters)] in Prism 7.0. Each PRNA was done in duplicate. Significance between neutralizing IC 50 values for each EMV and the irrelevant IgG control virus was calculated using 2-way ANOVA. The fold change in the mAb PRNA IC 50 was calculated by dividing the EMV IC 50 by the IC 50 of irrelevant IgG control virus B.
Mouse lethal dose. The 50% mouse lethal dose (mLD 50 ) for each EMV was determined using female BALB/c mice (at 6 to 8 weeks of age; Jackson Laboratory) in accordance with protocols approved by the Institutional Animal Care and Use Committee at the Icahn School of Medicine at Mount Sinai. Each virus was diluted from 10 5 to 10 1 in 1Â PBS, and 3 mice per dilution were infected (50 mL per mouse). Weight loss and survival were monitored daily for 14 days postinfection. Mice that lost more than 25% of their initial body weight were euthanized.